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2-Adrenergic-mediated tubular NO production
inhibits thick ascending limb chloride absorption
Hypertension and Vascular Research Division, Henry Ford Hospital, Detroit, Michigan 48202
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Stimulation
of 
2-adrenergic receptors inhibits transport in various
nephron segments, and the thick ascending limb of the loop of Henle
(THAL) expresses 2-receptors. We hypothesized that selective 2-receptor activation decreases NaCl
absorption by cortical THALs through activation of NOS and increased
production of NO. We found that the 2-receptor agonist
clonidine (10 nM) decreased chloride flux
(JCl) from 119.5 ± 15.9 to
67.4 ± 13.8 pmol · mm
1 · min1 (43%
reduction; P < 0.02), whereas removal of clonidine
from the bath increased JCl by 20%. When NOS
activity was inhibited by pretreatment with 5 mM
NG-nitro-L-arginine methyl ester,
the inhibitory effects of clonidine on THAL JCl
were prevented (81.7 ± 10.8 vs. 71.6 ± 6.9 pmol · mm1 · min1).
Similarly, when the NOS substrate L-arginine was deleted
from the bath, addition of clonidine did not decrease THAL
JCl from control (106.9 ± 11.6 vs.
132.2 ± 21.3 pmol · mm1 · min1). When we
blocked the 2-receptors with rauwolscine (1 µM), we found that the inhibitory effect of 10 nM clonidine on THAL
JCl was abolished, verifying that
2, rather than I1, receptors mediate the
effects of clonidine in the THAL. We investigated the mechanism of NOS
activation and found that intracellular calcium concentration did not
increase in response to clonidine, whereas pretreatment with 150 nM
wortmannin abolished the clonidine-mediated inhibition of THAL
JCl, indicating activation of
phosphatidylinositol 3-kinase and the Akt pathway. We found that
pretreatment of THALs with 10 µM LY-83583, an inhibitor of soluble
guanylate cyclase, blocked clonidine-mediated inhibition of THAL
JCl. In conclusion, 2-receptor stimulation decreases THAL JCl by increasing NO
release and stimulating guanylate cyclase. These data suggest that
2-receptors act as physiological regulators of THAL NO
synthesis, thus inhibiting chloride transport and participating in the
natriuretic and diuretic effects of clonidine in vivo.
nitric oxide synthase; clonidine; kidney
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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CLONIDINE IS AN
ANTIHYPERTENSIVE agent that acts through stimulation of
central 2-adrenergic receptors (34),
thereby inhibiting peripheral sympathetic tone and also markedly
affecting renal function (44). In vivo, clonidine
infusions have been associated with an increase in both sodium and
water excretion (15, 42, 46). The effects on urinary
output have been ascribed to activation of 2-adrenergic
receptors (22) both on the renal vasculature and along the
nephron (3).
2-Adrenergic receptors have been shown to mediate
inhibition of nephron transport in vitro. Rouse et al.
(43) demonstrated that direct 2-adrenergic
receptor activation inhibits proximal convoluted tubular fluid
absorption. Similarly, in the isolated cortical collecting duct,
2-adrenergic receptor activation inhibits vasopressin-stimulated hydroosmotic water permeability (10, 24) and amiloride-sensitive sodium reabsorption
(42). However, presently we know of no data directly
evaluating the effect of 2-adrenergic receptor
activation on the thick ascending limb of the loop of Henle (THAL).
Previous studies have demonstrated that NO plays an important role in the control of renal sodium excretion both in vivo (24) and in vitro (40, 45). We recently reported that THAL chloride absorption is directly inhibited by endogenously produced NO (36), and that eNOS mediates this response (37). However, the physiological regulation of tubular NOS is poorly understood.
2-Adrenergic receptors stimulate NO release in the
vascular endothelium. Blocking 2-adrenergic receptors
(2) enhances the vasoconstriction caused by the
sympathetic neurotransmitter norepinephrine, and
2-adrenergic receptor-induced vasodilatation is
sensitive to NOS inhibition (48). Furthermore, inhibition of the vasodilator effects of clonidine by
NG-monomethyl- L-arginine
(L-NMMA) can be overcome by adding the substrate for NOS,
L-arginine (39). Taken together, these
findings suggest that endothelial 2-adrenergic receptor
activation stimulates NOS and increases NO production.
The THAL expresses 2-adrenergic receptors (29,
55) and produces NO (36); however, it is not clear
whether 2-adrenergic receptor activation can inhibit
transport via an NO-dependent mechanism. We hypothesized that the
2-adrenergic receptor agonist clonidine decreases sodium
chloride absorption in the THAL by activating
2-adrenergic receptors, stimulating NOS, and increasing production of endogenous NO. Our findings indicate that clonidine inhibits chloride absorption in isolated perfused THALs by activation of 2-adrenergic receptors, acting through a
NOS-dependent mechanism via activation of phosphatidylinositol 3-kinase
(PI3K). Thus 2-adrenergic receptors may function as a
physiological regulator of THAL NOS activity.
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MATERIALS AND METHODS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Preparation of isolated nephron segments. Cortical THALs were obtained from male Sprague-Dawley rats, weighing 120-150 g (Charles River Breeding Laboratories, Wilmington, MA), which had been maintained on a diet containing 0.22% sodium and 1.1% potassium (Purina, Richmond, IN) with water ad libitum for at least 5 days. On the day of the experiment, rats were anesthetized with ketamine (100 mg/kg body wt ip) and xylazine (20 mg/kg body wt ip), and the abdominal cavity was opened to expose the kidney. The kidney was bathed in ice-cold saline and removed. Coronal slices were placed in oxygenated physiological saline at 12°C. Cortical THALs were dissected from medullary rays in the same solution under a stereomicroscope.
THAL perfusion.
THALs (0.5-0.9 mm) were transferred to a temperature-regulated
chamber and perfused between concentric glass pipettes at 37°C, as
described previously (36). The composition of the
basolateral bath and perfusate (in mmol/l) was 114 NaCl; 25 NaHCO3; 2.5 NaH2PO4; 4 KCl; 1.2 MgSO4; 6 alanine; 1 Na3 citrate; 5.5 glucose; 2 Ca lactate2 and 5 raffinose. In addition, a concentration
of L-arginine (4 µM) approximating the Michaelis-Menten
constant (Km) for eNOS (53) was
included in the bath and perfusate solutions, unless otherwise
indicated. Clonidine, the NOS inhibitor
NG-nitro-L-arginine methyl ester
(L-NAME), the NOS substrate L-arginine, the
2-adrenergic receptor antagonist rauwolscine, and the
PI3K inhibitor wortmannin were all purchased from Sigma (St. Louis, MO). The soluble guanylate cyclase inhibitor LY-83583 was purchased from Biomol (Plymouth Meeting, PA). The solution was bubbled with 5%
CO2-95% O2 before and during the experiments.
The pH of the bath was 7.4 and the osmolality of the bath solution was
290 ± 3 mosmol/kgH2O, as measured by freezing-point
depression. The basolateral bath was exchanged at a rate of 0.5 ml/min,
and tubules were perfused at 5 to 10 nl/min. Time-control studies were
conducted for each protocol to determine the stability of tubular transport.
Net chloride flux.
Chloride concentrations were determined in samples of perfusate and
collected fluid using a previously described fluorometric technique
(14). Because chloride reabsorption was not accompanied by
significant fluid reabsorption, net chloride flux
(JCl) was calculated according to the formula
![<IT>J</IT><SUB>Cl</SUB><IT>=</IT>PR([Cl]<SUB>o</SUB><IT>−</IT>[Cl]<SUB>I</SUB>)](/content/vol281/issue4/fulltext/F679/img001.gif)
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2-receptors in
the kidney for clonidine is well established and has been found to be
~2 nM (47). Therefore, to selectively activate
2-receptors, we used 10 nM clonidine in all protocols
evaluating the effects on THAL transport.
Experimental protocols.
We first tested the effects of 2-adrenergic receptor
agonists and antagonists on THAL JCl. In these
protocols, after a 20-min equilibration period, three basal
measurements were performed (control period). Then, clonidine, an
2-adrenergic receptor agonist, or rauwolscine, an
2-receptor antagonist, was added to the bath. Twenty
minutes later, three additional collections were made (experimental period).
2-adrenergic receptor stimulation with
clonidine, using two strategies. First, we measured the
JCl response to clonidine in the presence of NOS
inhibition. In this protocol, 5 mM L-NAME was present in
the bath solution throughout the experiment. We have previously
reported that 5 mM L-NAME alone does not significantly alter THAL JCl (36). After a 20-min
equilibration period, three basal measurements were performed (control
period). Clonidine was then added to the bath along with
L-NAME. After 20 min, three additional collections were
made (experimental period). Second, we evaluated the necessity of the
substrate for NOS, L-arginine, in the response of THALs to
clonidine. Thus in this protocol, L-arginine was omitted
from the bath and perfusion solutions. After a 20-min equilibration
period, three basal measurements were collected (control period).
Clonidine was then added to the bath solution, and after 20 min, three
additional collections were made (experimental period).
We evaluated the signaling cascade of NO in the response of THALs to
selective 2-adrenergic receptor stimulation. First, we
measured the JCl response in the presence of an
inhibitor of soluble guanylate cyclase, LY-83583 (10 µM). In this
protocol, LY-83583 was present in the bath solution throughout the
experiment. We have previously reported that 10 µM LY-83583 alone
does not significantly alter THAL JCl
(35). After a 20-min equilibration period, three basal
measurements were performed (control period). Clonidine was then added
to the bath along with LY-83583. After 20 min, three additional
collections were made (experimental period).
We tested the specificity of clonidine's effects on THAL
JCl by pretreating tubules with a selective
2-adrenergic receptor antagonist (rauwolscine; 1 µM).
After a 20-min equilibration period with rauwolscine in the bath, we
took three basal measurements (control period). We then added clonidine
(10 nM) to the bath along with the receptor antagonist. After a 20-min
equilibration period, three additional collections were performed
(experimental period).
Finally, we evaluated the mechanism of NOS activation in the response
of THALs to selective 2-adrenergic receptor stimulation with clonidine, using two strategies. First, we examined the effects of
clonidine on intracellular calcium concentration using a ratiometric fluorescent indicator technique (20). Briefly, tubules
were isolated, perfused, and superfused as described above and
incubated with 5 µM fura 2-AM (Molecular Probes, Eugene, OR) for
1 h. After washing for 30 min, basal intracellular calcium
concentration was determined. Excitation of fluorophores was performed
below 400 nM, and fluorescent emission was detected at greater than 510 nM. After 5 min of stable basal recording, tubules were exposed to 10 nM clonidine, and the response was recorded. We calibrated the maximum
and minimum calcium concentrations for each tubule by using 10 µM
4-Br-A23187 and 5 mM EGTA, respectively. Wavelength intensities and
ratios were sampled every 20 s by use of the MetaFluor imaging
software (Universal Imaging, West Chester, PA).
Second, we measured the JCl response in the
presence of an inhibitor of the PI3K/Akt pathway, wortmannin. In this
protocol, wortmannin (150 nM) was present in the bath solution
throughout the experiment. After a 20-min equilibration period, three
basal measurements were performed (control period). Clonidine was then added to the bath along with wortmannin. After 20 min, three additional collections were made (experimental period).
Statistics. Experimental results are expressed as means ± SE. Data were evaluated with Student's paired t-test. The criterion for statistical significance was P < 0.05 in all experiments.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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The THAL contains active NOS, and endogenously produced NO
inhibits THAL transport (36). Others have reported that
2-adrenergic receptors stimulate NO release
(39). Thus, we first evaluated the response of isolated
perfused THALs to clonidine, a selective 2-adrenergic
receptor agonist. Figure 1 illustrates
the effect of clonidine (10 nM) on JCl in seven
isolated THALs. During the control period, tubules absorbed chloride at
a rate of 119.5 ± 15.9 pmol · mm1 · min1. After 10 nM clonidine was added to the bath, tubules absorbed chloride at a rate
of 67.4 ± 13.8 pmol · mm1 · min1.
Perfusion rates did not differ during the two periods, and time controls showed no reduction in chloride absorption over a 2-h period.
Thus 10 nM clonidine inhibited THAL JCl by
43.3 ± 9.1% (P < 0.02).
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To verify that the reduction in transport was not secondary to any
cytotoxic effects of clonidine, we evaluated the ability of cortical
THALs to increase JCl after recovery from
clonidine exposure. Figure 2 depicts the
effects of removing 10 nM clonidine from the bath. In the presence of
10 nM clonidine, tubules absorbed chloride at a rate of 105.5 ± 11.8 pmol · mm1 · min1.
Thirty minutes after we removed clonidine from the bath,
JCl increased significantly to a rate of
125.4 ± 14.9 pmol · mm1 · min1
(20.3 ± 7.3%; P < 0.05; n = 7).
These findings indicate that the reductions in
JCl we observed in response to 10 nM basolateral clonidine administration were not secondary to cytotoxic effects.
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To test whether clonidine inhibits THAL JCl
through a combination of NOS activation and increased NO production, we
examined the effect of L-NAME on clonidine's ability to
inhibit JCl (Fig. 3). In the presence of 5 mM
L-NAME, tubules absorbed chloride at a rate of 81.7 ± 10.8 pmol · mm1 · min1
(n = 7). When we added 10 nM clonidine to the bath,
THAL chloride absorption did not change significantly from the basal
rate (71.6 ± 6.9 pmol · mm1 · min1). Because
we have previously found that 5 mM L-NAME alone does not
significantly alter JCl (36), these
findings suggest that clonidine inhibits THAL transport via a
NOS-dependent mechanism.
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Because NOS inhibition abolished the ability of clonidine to inhibit
THAL transport, we next evaluated the role of exogenous L-arginine, the substrate for NOS, in clonidine's effects
by measuring the effect of clonidine on THAL JCl
in the absence of exogenous L-arginine (Fig.
4). During the control period, tubules
absorbed chloride at a rate of 106.9 ± 11.6 pmol · mm1 · min1. After 10 nM clonidine was added to the bath, chloride absorption was not
significantly different from the basal rate (132.2 ± 21.3 pmol · mm1 · min1;
n = 6). Thus removing the substrate for NOS prevented
the reduction in THAL chloride absorption induced by 10 nM clonidine.
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The effects of NO in many tissues are mediated by the stimulation of
guanylate cyclase and increased cGMP (32). To examine whether 2-adrenergic receptor-mediated NO production
follows a similar signaling cascade, we next examined the effects of an inhibitor of soluble guanylate cyclase, LY-83583, on clonidine's ability to inhibit THAL chloride absorption. In the presence of 10 µM
LY-83583, tubules absorbed chloride at a rate of 66.1 ± 11.9 pmol · mm1 · min1
(n = 6). When we added 10 nM clonidine to the bath,
THAL chloride absorption did not change significantly from the basal
rate (53.5 ± 3.0 pmol · mm1 · min1; 6.8 ± 16.4%). Because we have previously reported that 10 µM LY-83583
alone does not significantly alter JCl
(35), taken together, the present findings suggest that
clonidine inhibits THAL transport via activation of NOS, increased NO
production, and stimulation of soluble guanylate cyclase.
We have reported that the endothelial isoform of NOS mediates the
inhibitory effects of L-arginine in the THAL
(37). Other investigators have demonstrated that
activation of endothelial NOS is calcium dependent (53).
Therefore, we next examined the intracellular calcium response of
isolated perfused THALs to activation of 2-adrenergic
receptors with clonidine. Intracellular calcium concentration increased
only 22 ± 4% from the basal value of 114.5 ± 14.8 nM in
response to 10 nM clonidine. These findings indicate that
2-adrenergic receptors do not likely activate THAL NOS
by increasing intracellular calcium concentration. Thus we explored alternative mechanisms for the activation of NOS by
2-adrenergic receptors.
Previous studies have demonstrated that eNOS may also be activated
through a calcium-independent pathway via stimulation of PI3K and
phosphorylation of the serine/threonine kinase Akt (12, 13). Therefore, we next examined the possibility that
2-adrenergic receptors stimulate THAL NOS and increase
NO production through activation of PI3K. Figure
5 depicts the effects of 10 nM clonidine on five tubules pretreated with the PI3K inhibitor wortmannin (150 nM).
During the control period, tubules absorbed chloride at a rate of
108.6 ± 13.1 pmol · mm1 · min1. After 10 nM clonidine was added to the bath, THAL chloride absorption did not
change significantly from the basal rate (98.9 ± 10.7 pmol · mm1 · min1). In a
separate series of experiments, addition of 150 nM wortmannin alone did
not significantly alter JCl from control
(82.6 ± 15.1 vs. 75.1 ± 7.9 pmol · mm1 · min1;
n = 5). Taken together, these findings suggest that
clonidine stimulates THAL NOS activity primarily through a
PI3K-mediated pathway, whereas PI3K is not constitutively active under
basal conditions.
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Other investigators have reported that clonidine stimulates
I1-type imidazoline receptors (8) as well as
2-adrenergic receptors. To verify that the effects of
clonidine are specifically mediated by 2-adrenergic
receptors, we examined the effect of the 2-adrenergic
receptor antagonist rauwolscine on clonidine's ability to inhibit THAL
chloride absorption (Fig. 6). In the
presence of 1 µM rauwolscine, tubules absorbed chloride at a rate of
106.8 ± 24.4. pmol · mm1 · min1
(n = 6). After 10 nM clonidine was added to the bath,
THAL chloride absorption did not change significantly from the basal
rate (93.4 ± 18.7 pmol · mm1 · min1). Control
experiments demonstrated that rauwolscine alone did not alter THAL
JCl from control (101.1 ± 11.9 vs.
92.4 ± 14.3 pmol · mm1 · min1;
n = 6). Thus clonidine inhibits THAL
JCl specifically via 2-adrenergic receptors.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Our data show that clonidine reversibly inhibits
JCl by isolated perfused THALs that can be
blocked by a competitive inhibitor of NOS, the removal of the substrate
for NOS, L-arginine, or the inhibition of soluble guanylate
cyclase. Moreover, a selective 2-receptor antagonist,
rauwolscine, and wortmannin, an inhibitor of PI3K, were able to block
clonidine-induced decreases in JCl. As a whole,
these findings suggest that clonidine inhibits THAL transport by
1) enhancing endogenous NOS activity, 2)
releasing NO, and 3) stimulating soluble guanylate cyclase
via activation of 2-adrenergic receptors and PI3K. This
suggests that 2-adrenergic receptors are physiological
regulators of THAL NOS.
2-Receptors inhibit THAL transport.
The present studies demonstrate that selective activation of
2-adrenergic receptors inhibits THAL
JCl, thus supporting the findings of other
investigators who demonstrated 2-mediated inhibition of
transport in other nephron segments. Bello-Reuss (4)
demonstrated that addition of the adrenergic neurotransmitter
norepinephrine to the bath increased fluid absorption in microperfused
proximal convoluted tubules. The stimulatory effects of norepinephrine were abolished by pretreatment with the 
-antagonist propranolol, which in turn unmasked significant inhibition of fluid absorption in
response to norepinephrine, an effect that may have been mediated by
2-adrenergic receptor activation. Rouse et al.
(43) examined this phenomenon directly and demonstrated
that selective 2-adrenergic receptor stimulation with
clonidine decreased fluid absorption in the isolated perfused proximal tubule.
2-Adrenergic receptors also inhibit fluid absorption,
and depending on the species, sodium absorption, in the collecting duct. Krothapalli et al. (24) showed that
2-adrenergic receptor activation inhibits arginine
vasopressin (AVP)-induced osmotic water permeability in rabbit cortical
collecting ducts, while Chen et al. (10) reported that
clonidine reversibly inhibited both AVP-dependent osmotic water
permeability and lumen-to-bath sodium flux in the rat cortical
collecting duct. Chen et al. (10) were able to block these
effects by pretreatment with a selective 2-antagonist,
indicating that clonidine's effects were indeed mediated by activation
of 2-adrenergic receptors. Thus our present findings
support an inhibitory role for 2-adrenergic receptors in
the THAL, where clonidine decreases sodium chloride absorption through
activation of 2-adrenergic receptors.
We believe these are the first in vitro data showing that clonidine,
acting via 2-adrenergic receptors, stimulates NO
production to inhibit transport in any nephron segment. Moreover, they
support previous in vivo data suggesting that clonidine directly
influences urinary sodium excretion. In previous studies, intrarenal
administrations of clonidine increased free water clearance, and to a
lesser extent, sodium excretion (3, 41, 46). The diuretic
and natriuretic responses were observed independently of changes in
glomerular filtration rate or renal perfusion pressure
(5). Taken together, these data suggest that clonidine,
acting through activation of 2-adrenergic receptors,
stimulates the production of NO, which in turn increases urinary sodium
excretion by a direct tubular effect.
Evidence indicating that renal 2-adrenergic receptors
stimulate NO production has been previously assayed by measuring the conversion of the NOS substrate L-arginine to its
byproduct, L-citrulline (49). That same study
demonstrated that selective blockade of medullary
2-adrenergic receptors with rauwolscine reduced renal NOS activity (and hence L-citrulline accumulation) and
instigated arterial hypertension to otherwise subpressor intravenous
infusions of norepinephrine (49). The renal hemodynamic
response following rauwolscine was mimicked by renal interstitial
infusion of L-NAME, suggesting that renal medullary
2-adrenergic receptors act through stimulation of NO
production. The THAL comprises a significant portion of the outer
medulla (9), and the present study demonstrates that
clonidine's ability to inhibit THAL transport is sensitive to both
rauwolscine and L-NAME. Thus the
2-receptor-mediated renal NO production observed in the
previous whole-animal studies may have been derived in part from the THAL.
Some previous investigators have been unable to observe any effect of
2-adrenergic receptors on transport in the THAL
(3). The bulk of the earlier studies were concerned with
the effects of efferent renal nerve stimulation or direct intrarenal
infusions of 2-adrenergic receptor agonists on renal
function in the whole animal (11).
2-Adrenergic receptor activation affects intrarenal neurotransmitter release (21), endothelial NO production
(48), renal hemodynamics (15), and proximal
tubular transport (16, 33, 43). Therefore, direct effects
of 2-adrenergic receptor-mediated effects of renal
sympathetic nerve stimulation on THAL transport in the whole kidney may
be obscured during intravenous or intrarenal infusions of antagonists.
We believe the isolated perfused THAL preparation obviates many of the
potentially confounding influences of simultaneous activation of
2-adrenergic receptors in other renal cell types and
thus clarifies their role, at least in this specific nephron segment.
2-Receptors and stimulation of NOS.
The specific 2-adrenergic receptor isoform(s) by which
clonidine stimulates THAL NOS activity is presently unknown. However, we found that clonidine-mediated inhibition of THAL
JCl was sensitive to the selective antagonist
rauwolscine, suggesting that this response is dependent on
2-adrenergic, rather than I1-imidazoline, receptor activation. Bockman et al. used differential receptor binding
affinities to 2-agonists and antagonists and implicated the 2A-, 2C- (6), and
2D- (7) adrenergic receptor subtypes in
stimulation of endothelial NO production. Furthermore, a recent study
(55) using RT-PCR of microdissected nephron segments
demonstrated expression of all known 2-receptor subtypes
in the rat THAL. Thus multiple isoforms of 2-adrenergic
receptors may be coupled to NO production in the THAL. Additional
studies are necessary to determine which specific
2-adrenergic receptor subtype mediates the stimulation
of THAL NOS activity.
2-adrenergic receptors stimulate
NO production in the THAL is presently uncertain. However, our data showing abolition of clonidine-mediated inhibition of THAL
JCl by L-NAME and substrate
deprivation indicate that this response is dependent on activation of
NOS and increased NO production. Each of the three isoforms of NOS has
been localized to the THAL (1, 28, 30, 51). The
constitutively expressed NOS isoforms [i.e., endothelial (eNOS) and
neuronal (nNOS)] have traditionally been thought to require increased
intracellular calcium for activation, whereas inducible NOS (iNOS) does
not but is dependent on substrate availability (53). We
have recently reported that eNOS mediates the inhibitory effects of
exogenous L-arginine on THAL JCl
(37), and others have found that
2-adrenergic receptor activation increases intracellular
calcium concentrations (17). Therefore we initially investigated the mechanism of 2-adrenergic receptor
activation of THAL NOS activity by examining the intracellular calcium
response to clonidine. To our surprise, intracellular calcium
concentration was unaffected by exposure to clonidine. Because the
Km of NOS for calcium is 200 nM
(19), increasing intracellular calcium from the basal
concentration of 115 to 140 nM would be sufficient to increase NOS
activity from 35 to 40% of its maximal rate. Given this very modest
response of intracellular calcium, we evaluated an alternative
signaling pathway for the activation of THAL NOS.
Recent findings have demonstrated that eNOS can be activated through
calcium-independent mechanisms. Dimmeler et al. (12) have
shown that activation of PI3K and stimulation of the serine/threonine protein kinase Akt can stimulate eNOS. This signaling pathway and NO
production are sensitive to the fungal derivative wortmannin. In
addition, others have reported that 2-adrenergic
receptor-mediated responses are sensitive to inhibition of PI3K
(52). Wortmannin pretreatment abolished the
inhibitory effects of clonidine on THAL
JCl, indicating that 2-adrenergic
receptors are likely coupled to the activation of THAL NOS through
stimulation of PI3K and increased Akt activity. To our knowledge, this
is the first report of NOS activation being mediated through activation
of the PI3K/Akt-signaling pathway in any tubular segment.
Possible physiological interactions and implications. The THAL is critical in the control of sodium excretion, absorbing ~25% of the filtered sodium chloride load (23), and the present studies demonstrate that clonidine inhibits THAL JCl. Because sodium is required for chloride transport across the apical membrane, and the Na-K-ATPase drives the Na-K-2Cl cotransporter, sodium reabsorption must accompany THAL chloride reabsorption (31). Therefore, clonidine may be expected to increase urinary sodium chloride excretion, with all other neurohumoral controllers of renal function remaining constant.
Because we have shown that clonidine stimulates THAL2-adrenergic receptors, and in turn, NOS activity, there
may be additional effects on glomerular hemodynamics. Under normal
conditions, the inhibition of THAL JCl would
increase sodium chloride delivery to the macula densa and would result
in tubuloglomerular feedback (TGF) that would reduce the delivery of
sodium chloride from the THAL to a normal value. NO has been
demonstrated to blunt the TGF response (50), whereas
inhibitors of NOS augment the TGF response (54). NO has
been predicted to have a half-life of 5 s (27) and
has a diffusion constant of 3,300 µm2/s
(26). Given these parameters, it is possible that NO
produced by the THAL may be carried via the tubular fluid downstream to the macula densa. Alternatively, our laboratory has recently shown that
NO produced by the macula densa directly inhibits TGF rather than via
diffusion to the afferent arteriole (38). Thus
clonidine-stimulated NO production by the THAL or the macula densa
itself may reset the TGF mechanism, reducing its efficiency and
promoting sodium chloride excretion.
The THAL is also impermeable to water, and absorption of salt by this
nephron segment both establishes and maintains the hypertonic medullary
solute gradient and also generates dilute tubular fluid (18,
31). To this end, acute clonidine-mediated reductions in sodium
chloride absorption would be expected to reduce the diluting ability of
the THAL. Exposure of the THAL to 2-adrenergic stimulation would then decrease the corticomedullary solute gradient and decrease the kidney's ability to concentrate urine. The widely observed phenomena of dilute urine production and increased free water
clearance due to clonidine in vivo have generally been attributed to
2-receptors antagonizing the effects of vasopressin in
the collecting duct (15, 24). However, the high transport
rates and water-impermeant nature of the THAL are the cardinal
attributes of the kidney that allow selective water abstraction in the
downstream nephron. Therefore, the inhibition of sodium chloride
absorption in the THAL may provide a mechanistic basis for the previous
in vivo findings of clonidine having potent effects on urinary
concentrating ability and hence promoting water excretion.
Conclusion.
We found that clonidine inhibited chloride absorption by the isolated
perfused THAL via activation of 2-adrenergic receptors. This inhibition was abolished by L-NAME and required the
substrate for NOS activity, L-arginine. The response was
specifically mediated by 2-adrenergic receptors, because
pretreatment with the selective antagonist rauwolscine prevented the
effects of clonidine. These findings indicate that the rat THAL
responds to 2-adrenergic receptor activation by
increasing production of NO, which then inhibits transport via an
autocrine mechanism. Thus 2-adrenergic receptors may be
physiological regulators of THAL NOS activity, and the inhibitory
effects of clonidine on THAL chloride absorption may partially explain
its ability to increase urinary sodium and water excretion in vivo.
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ACKNOWLEDGEMENTS |
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This work was conducted during the tenure of an American Heart Association Fellowship Grant awarded to C. F. Plato. It was supported by National Heart, Lung, and Blood Institute Grant HL-28982 awarded to J. L. Garvin.
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FOOTNOTES |
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Address for reprint requests and other correspondence: J. L. Garvin, Henry Ford Hospital, Hypertension and Vascular Research Division, 2799 W. Grand Blvd., Detroit, MI 48202.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 12 December 2000; accepted in final form 25 May 2001.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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